Four homologous 80 amino acid peptides or saposins are encoded in tandem and in-frame by the prosaposin locus. These saposins or activator proteins are essential for normal function of several hydrolases involved in the glycosphingolipid/sphingolipid (GSL/SL) catabolic pathway in lysosomes. Consequently, the flux in this GSL/SL pathway and the production of resultant bioactive end products (i.e., lysosphingolipids) are controlled by the prosaposin locus. We seek to characterize and elucidate the molecular enzymology and processing of this unique multifunctional protein in complex GSL metabolism. The studies are designed to determine the structural correlates of prosaposin derived saposin activities to gain greater insight into the nature of their function. Immunofluorescence studies with our specific prosaposin, and saposin A, B, C and D antibodies will be used to characterize the landscape of temporal/spatial expression of prosaposin in the mouse and selected human tissues. Based on preliminary immunofluorescence, western blot and in situ hybridization studies, the prosaposin locus is differentially expressed in various tissues. Such distinct expression in tissues suggests alternative requirements and controls in different cell types. The tissue specific proteolytic and/or glycosidic processing events of prosaposin to mature saposins A, B, C and D will be elucidated via pulse-chase processing studies in selected tissues to provide a basis for understanding the modulation of GSL and SL flux through the lysosomal pathway. In addition, our knowledge of the nature of saposin-enzyme-lipid interactions is rudimentary. The proposed studies focus on saposins C and B as prototypes for detailed analyses since their mechanisms of interactions with their respective enzymes appear distinct. These saposins can be expressed in active forms in E. coli and subjected to extensive site directed mutagenesis. The conservation of cysteines in the saposins will permit the use of chimeric proteins composed of parts of two saposins for localizing important regions for binding, activation and specificity of these prototype saposins. The expanding central role of SLs, sphingoid bases and ceramides as second messengers, cellular growth modulators and mediators of cell-cell interaction, circumscribes the need for detailed understanding of the metabolism of these compounds. The significance of the proposed studies relates to the elucidation of the control at the enzymatic and tissue levels of critical GSL and SL pathways that have major effects on the modulation of sphingolipid hydrolases and influence on the expression of lysosomal storage diseases.